Loss of inner ear sensory cells is a major cause of permanent hearing loss. Our long-term goal is to provide a treatment option for patients suffering from hair cell loss by in vitro selection and characterization of progenitor cells that ultimately can be used for cell-replacement therapy of the damaged inner ear. We will select and characterize inner ear progenitor cells from two sources. First, we will optimize the isolation of progenitor cells derived from the adult mouse utricle (Aim 1). We have found in preliminary studies that the mouse utricle contains cells that display defining characteristics of progenitor cells, that these cells can be maintained in culture for several months, and that these cells can differentiate into cells that express immunological markers indicative of hair cells. A second source for progenitor cells is embryonic stem (ES) cells. In pilot studies, we have generated from mouse ES cells a population of cells that displayed expression of markers genes indicative of the developing inner ear. Our second Aim is to establish in vitro conditions that enable us to routinely guide ES cell derivatives towards a progenitor cell phenotype that can be differentiated into mature inner ear cell types, which can be functionally characterized by in vitro and in vivo studies. In Aim 3, we propose testing whether expression of the transcription factor Math1 in stem cells, including the progenitor cells generated with the experiments proposed in this application, can lead to increase in the number of generated hair cells. Finally, we will perform basic analyses of Features of progenitor cell-derived hair cell-like cells. We will test whether in vitro-generated hair cell-like cells do selectively label with the styryl dye FM1-43 and whether they display electrophysiological features of native hair cells (Aim 4A). In Aim 4B, we will generate major inner ear cell types in vivo by grafting presumptive progenitor cells into the developing chicken inner ear. [unreadable] [unreadable]